Delayed Development of Fear-Potentiated Startle in Rats
Copyright 1994 by the American Psychological Association, Inc.
Behavioral Neuroscience
0735-7044/94/S3.00
1994, Vol. 108, No. 1,69-80
http://ishare.iask.sina.com.cn/f/33945368.html
Pamela S. Hunt, Rick Richardson, and Byron A. Campbell
Pamela S. Hunt, Rick Richardson, and Byron A. Campbell, Department of Psychology, Princeton University.
This research was supported by National Institute of Mental Health Grants MH01562 and MH49496 to Byron A. Campbell and National Institutes of Child Health and Human Development Postdoctoral Grant HD07694 to Pamela S. Hunt.
Correspondence concerning this article should be addressed to Pamela S. Hunt, Department of Psychology, Princeton University, Green Hall, Princeton, New Jersey 08544-1010. Electronic mail may be sent to pshunt@pucc.princeton.edu.
The developmental emergence of fear-potentiated startle was examined in rats ranging in age from 16 to 75 days. In Experiment 1, a pure tone served as the conditioned stimulus (CS) and an acoustic startle pulse served as the unconditioned stimulus (US) for fear conditioning. Fear-potentiated startle by the tone CS was observed in rats 23 days of age and older but not in rats 16 days of age. In Experiment 2, a light served as the CS. Rats 30 days of age and older showed fear-potentiated startle, whereas 23-day-old rats did not. The final experiment demonstrated that another behavioral index of fear, stimulus-elicited freezing, was observed earlier in development than fear-potentiated startle, confirming the effectiveness of the training procedure for conditioning fear. The results suggest that fear-potentiated startle is a relatively late-emerging response system, parallelling the development of conditioned autonomic changes (e.g., heart rate) rather than that of freezing.
Fear-potentiated startle is a commonly used measure of fear conditioning (Brown, Kalish, & Farber, 1951; Davis & Astrachan, 1978; Hamm, Greenwald, Bradley, Cuthbert, & Lang, 1991; Lang, Bradley, & Cuthbert, 1990; Leaton & Borszcz, 1985). For example, when rats are presented with a startle-eliciting stimulus, typically a brief, intense auditory stimulus, they respond behaviorally with a whole-body jerk, referred to as the startle reflex. When the startle stimulus is preceded by a cue that evokes fear, such as a light that has been paired with shock, the startle response is greater than when the startle stimulus is presented alone. This enhancement in startle responding is termed fear-potentiated startle, and it has been extensively used as a tool for examining the pharmacological and neuroanatomical bases of conditioned fear (Cassella & Davis, 1985; Davis, 1979, 1984, 1986, 1992a, 1992b; Davis, Hitchcock, & Rosen, 1992; Hitchcock & Davis, 1986, 1987, 1991; Rosen & Davis, 1988; Sananes & Davis, 1992). The purpose of the following experiments was to investigate the emergence of fear-potentiated startle during development, using auditory and visual stimuli as conditioned stimuli (CSs).
Two disparate lines of evidence converge to make the ontogenetic study of fear-potentiated startle particularly interesting. First, Wecker and Ison (1986) showed that the magnitude of the startle response is highly correlated with the type of behavior occurring just prior to the elicitation of startle. In their study, adult rats were shown to exhibit a greater startle response when they were immobile prior to the presentation of a startle-eliciting noise burst than when they were active. The reduction in startle response magnitude during activity was particularly marked when the animals were engaged in consum-ing (eating or drinking), grooming, or sniffing behavior. This relation between activity and startle response magnitude has also been implicated in fear-potentiated startle. According to Leaton and Borszcz (1985), a CS that has been paired with an aversive stimulus elicits the species-specific response of freezing in the laboratory rat and that when that CS precedes a startle stimulus, the resulting immobility potentiates the startle response (see also Leaton & Cranney, 1990).
Although we could find no research on the correspondence between freezing and startle responding during development, several researchers have demonstrated that infant, as well as adult, rats show a decrease in activity in the presence of a CS that has been paired with shock (Campbell & Ampuero, 1985; Coulter, Collier, & Campbell, 1976; Mellon, Kraemer, & Spear, 1991; Moye & Rudy, 1985, 1987). For example, Moye and Rudy (1987) paired a tone CS with shock for preweanlings rats of different ages; when the tone was presented 24 hr later, it produced a substantial decrease in general activity in rats 15 days of age and older. It is a reasonable prediction then that animals as young as 15-16 days of age would show the potentiated startle effect given their disposition to respond with inactivity (freezing) to a tone that had been paired with shock.
A second line of evidence, however, suggests that potentiated startle may not be observed until much later in development. A number of investigators, working with other species such as the rabbit and cat, have shown that reflex sensitivity is altered by stimulation of the amygdala, particularly the central nucleus, from which concomitant changes in heart rate (HR) are also evoked. Whalen and Kapp (1991) reported that the amplitude of the rabbit's nictitating membrane reflex (NMR) was enhanced by stimulation of the central nucleus of the amygdala that also evoked bradycardia but that it was inhibited by stimulation of other regions of the amygdala that concomitantly evoked tachycardia. From these results, Whalen and Kapp (1991) proposed that heart rate change and reflex modification covaried, with heart rate decreases accompanying facilitation and heart rate increases accompanying inhibition of the magnitude of the response (see also Gary Bobo & Bonvallet, 1975; Gebber & Klevans, 1972; Kapp, Whalen, Supple, & Pascoe, 1992; Marks, Frysinger, Trelease, & Harper, 1983; Pascoe, Bradley, & Spyer, 1989; Schlor, Stumpf, & Stock, 1984). Although none of these researchers explicitly postulated a causal relation between HR changes and alterations in reflex amplitude, the findings that the two measures occur together suggest that they are somehow related.
Conditioned changes in HR have been documented in young rats when auditory and visual stimuli are paired with an aversive stimulus such as shock (Campbell & Ampuero, 1985) or a startle pulse (Richardson, Wang, & Campbell, 1993). However, conditioned heart rate responses emerge much later in development than behavioral measures of fear such as freezing or conditioned suppression of locomotion. Specifically, although there are numerous demonstrations of stimulus-elicited freezing to auditory and visual CSs as early as 15-17 days postnatal (Campbell & Ampuero, 1985; Coulter et al., 1976; Mellon et al., 1991; Moye & Rudy, 1985, 1987), conditioned changes in heart rate to auditory and visual stimuli are not observed until approximately 21 and 28 days of age, respectively (Campbell & Ampuero, 1985). Thus, if facilitation of the startle reflex is correlated with stimulus-elicited changes in heart rate (in this case attributable to conditioning), potentiated startle would not be expected to occur until about 21 days of age to auditory stimuli that had previously been paired with shock and not until 28 days of age to visual stimuli. These two lines of evidence lead to quite different predictions concerning the age at which fear-potentiated startle should first emerge during the course of development. If it is conditioned immobility (freezing) that is associated with enhanced startle magnitude, then fear-potentiated startle to auditory stimuli should emerge about 15 days of age and around 17 days of age to visual stimuli. Conversely, if conditioned changes in heart rate are critical predictors of startle modulation, then fear-potentiated startle should not be observed until approximately 21 and 28 days of age to auditory and visual stimuli, respectively.
Experiments 1A and IB
In the fear-potentiated startle paradigm, fear is typically conditioned off-line with shock as the unconditioned stimulus (US; Brown et al., 1951; Davis, 1984, 1992a, 1992b). However, Leaton and Cranney (1990) demonstrated that the startle pulse itself could be used as a US to establish fear to a visual CS. Using the startle pulse as a US has one particular advantage for conditioning fear to the CS over off-line shock in that it allows the experimenter to observe the acquisition of fear-potentiated startle trial by trial. Also, Richardson et al. (1993) demonstrated the effectiveness of the startle pulse as an aversive US for HR conditioning. With repeated pairings of a tone CS with the startle pulse, animals 21 days of age and older showed reliable conditioned bradycardia to the tone. However, 16- and 17-day-olds did not exhibit conditioned cardiac changes in this situation. These results are strikingly similar to those reported by Campbell and Ampuero (1985) with shock as the US.
The purpose of Experiment 1A was to study the ontogeny of fear-potentiated startle using an 80-dB, 1600-Hz tone as the CS and a 130-dB burst of white noise as the US. Three age groups of rats—16, 23, and 75 days—were selected to investigate the emergence of fear-potentiated startle during development.
Experiment 1A: Method
Subjects. The subjects were experimentally naive Sprague-Dawleyderived rats, bred and reared in the vivarium of the psychology department of Princeton University. On Day 2 after birth (day of birth = Day 0), litters were culled to 8 pups. Animals were either 16 (± 1), 23 (± 1), or 75 (± 15) days old at the time of testing. Purina Rat Chow and water were available in the home cage ad libitum. Both male and female rats were included in the 16- and 23-day-old groups (ns = 10), whereas only males were tested at 75 days of age (n = 1011). Animals tested at 16 and 23 days of age were maintained with their dams and littermates in 48.0-cm x 25.5-cm x 20.5-cm polycarbonate cages. No more than 2 rats from any given litter were assigned to each treatment group. Rats tested as adults were weaned when 23-26 days old and housed in groups of five with same-sex conspecifics in hanging wire cages. Adult animals were housed singly and handled daily 3 days prior to training. The vivarium was maintained on a 16:8 light-dark cycle, with light onset at 7 a.m.
Apparatus, Rats were trained and tested in a 16.3-cm x 8.5-cm x 8.5-cm plastic and stainless steel restraining cage (Coulbourn Instruments, Allentown, PA, Model EOS-15). The restraining cage was placed on a Coulbourn Instruments transducer platform (Models E45-11 and E45-15) that was placed in an Industrial Acoustics (Lodi, NJ) sound-attenuating chamber (IAC). The IAC contained a wall-mounted 7.5-W houselight, a floor-mounted JBL speaker (Model 2426H) for producing the startle stimulus, and a ceiling-mounted Jensen speaker (Model Jl 176) for producing the CS.
The auditory CS used in this research consisted of a 10-s, 1600-Hz, pulsating (2 pulses per second with a rise-fall time of 250 ms) pure tone of 80 dB as measured on the C scale of a Simpson (Elgin, IL) sound level meter (Model 886). The US was an acoustic startle stimulus consisting of a 100-ms, 130-dB burst of white noise, with an instantaneous rise-fall time.
The startle response was recorded using a Coulbourn Instruments Startle Response System. The system consisted of two transducer platforms that produced electrical signals proportional to the force applied to them. One platform with a 1-lb (0.454-kg) maximum force limit was used for the 16- and 23-day-old rats and one with a 5-lb (2.27-kg) maximum was used for the adult rats. The analog output of each platform was converted to a digital score for each millisecond of a 200-ms period that began with the onset of the startle stimulus. From these data, we determined the peak startle response. The sensitivity of the startle recording equipment was adjusted to produce peak force outputs in the middle of the platform's sensitivity for each age. The range for both platforms was 0-255 mV, and the average peak outputs on the first 3 trials of the training session for the three ages were 132 mV for the 16-day-old rats, 126 mV for the 23-day-old rats, and 167 mV for the 75-day-old rats. Although the peak outputs of the adult rats were somewhat higher than that of the younger rats, they were still within a range that permitted assessment of the fear-potentiated startle response.
Procedure. Rats were removed from their home cages, randomly assigned to either a paired or explicitly unpaired group, weighed, and placed in the restraining cage, which was then positioned on the transducer platform inside the IAC chamber. Rats were trained and tested individually. All subjects were given a 15-min period of adaptation prior to the onset of training.
Training consisted of 4 CS-alone habituation trials, followed by either 20 paired or 20 unpaired trials. Animals in the paired group were given 20 CS-US presentations, in which the offset of the 10-s tone CS was paired with the 100-ms, 130-dB startle stimulus. The intertrial intervals (ITIs) ranged from 60 to 120 s. Unpaired animals received the same number of CSs and USs but in an explicitly unpaired fashion. The order of CS and US presentations was random, with the stipulation that not more than 3 of a given trial type could occur in succession, and the interstimulus intervals (ISIs) ranged from 30 to 60 s.
Figure 1. Mean (and standard error of the mean) startle responses (in millivolts) of 16-, 23-, and 75-day-old subjects in Experiment 1A. Rats were given paired or explicitly unpaired presentations of a tone conditioned stimulus (CS) with the startle pulse during fear conditioning and were tested for startle responding immediately after acquisition. For testing, animals were given the startle pulse presented alone (noise-alone trials) or preceded by the 10-s tone CS (tone-noise trials).
Immediately following completion of the training sequence, all of the animals were given 6 additional test trials with 60-120-s ITIs. On 3 of these trials, the startle pulse was presented alone (noise-alone trials), and on the other 3 trials the startle pulse was preceded by the 10-s tone CS (tone-noise trials). The order of test trials was randomly determined. Fear-potentiated startle was assessed by comparing the magnitude of the startle response on the tone-noise trials with that on the noise-alone trials.
Statistical analyses. The startle responses (analog-to-digital output, in millivolts) for each rat were averaged over blocks of two training trials for the acquisition phase. These data were analyzed separately for subjects of each age using a 2 (group) x 10 (trial block) mixed analysis of variance (ANOVA). The responses to the startle pulse during test for each subject were averaged over the three trials of each type (noise alone or tone-noise) and were analyzed separately for each age using a 2 (group) x 2 (trial type) mixed ANOVA. When appropriate, post hoc comparisons were made using Newman-Keuls tests (p = .05), and planned comparisons were conducted between groups with t tests (p = .05).
Experiment 1A: Results
Test data. Analyses of the data obtained during the test phase revealed that 23-day-oIds and adults exhibited fear-potentiated startle, whereas 16-day-olds did not. For the two older groups in the paired condition, the magnitude of the startle response was greater when the startle stimulus was preceded by the fear-eliciting CS than when it was presented alone. By contrast, the 16-day-old subjects responded equivalently to the startle pulse, regardless of training experience and test trial type. The mean startle response amplitudes for the test trials for the paired and unpaired groups are presented in Figure 1.
The 2 (group) x 2 (trial type) mixed ANOVA conducted on the test data for the 16-day-olds revealed no significant effects (all Fs < 1.0). The ANOVA conducted on the data from the 23-day-olds yielded a significant main effect of trial type, F(l, 18) = 7.58, p < .05. Planned comparisons confirmed that for the paired group, responding to the startle pulse was greater on the tone-noise trials than on the noise-alone trials. For the unpaired group, responding was the same on both trial types.
The ANOVA conducted on the adult test data revealed a significant main effect of trial type, F(l, 19) = 21.32, p < .01, as well as a significant Group x Trial Type interaction, F(l, 19) = 6.73, p < .05. Although the response to the startle stimulus was the same for the unpaired rats on both test trial types, the paired rats responded more on the tone-noise trials than on the noise-alone trials.
Acquisition data. The mean startle response amplitudes during the acquisition phase are presented in Figure 2. A 2 (group) x 10 (trial block) mixed ANOVA conducted on the data obtained from the 16-day-olds yielded a significant main effect of trial block, F(9, 162) = 4.90, p < .01. The main effect of group and the Group x Trial Block interaction were both nonsignificant (Fs < 1.0). The startle responses of both groups showed equivalent habituation over the course of the training session.
Figure 2. Mean startle responses (in millivolts) of 16-, 23-, and 75-day-old subjects during the acquisition phase of Experiment 1 A. Subjects were given either paired or explicitly unpaired presentations of a tone conditioned stimulus with the startle pulse unconditioned stimulus. Startle responses were averaged over blocks of 2 training trials.
was nonsignificant, F(9, 162) = 1.14,p > .05. Startle response amplitude habituated over the course of training for both groups, but the paired group showed consistently higher levels of responding than the unpaired group. The apparent difference between the initial levels of responding (Trial Block 1) was nonsignificant,F(l, 18) = 3.43,p > .05, whereas on Trial Block 10 the two groups were significantly different, F(l, 18) = 10.77,p < .01.
An analysis conducted on the acquisition data obtained from the adult subjects yielded significant main effects of group, F(l, 19) = 7.19,p < .05, and trial block, F(9,171) = 3.33,p < .01, as well as a significant interaction between group and trial block, F(9, 111) = 2.76,p < .05. All rats began training with a high level of responding to the startle stimulus, and this response habituated across the training episode in the unpaired group. For the paired group, responding was maintained at a high level throughout the training procedure.
Experiment 1A: Discussion
The results of this experiment suggest that fear-potentiated startle does not emerge ontogenetically until approximately 23 days of age, at least when an auditory stimulus serves as the CS and the startle pulse serves as the US for fear conditioning. After 20 pairings of the tone CS with the startle pulse US, 23-day-olds and adults showed exaggerated startle responses on the tone-noise test trials compared with the level of responding on the noise-alone trials. By contrast, there were no differences in startle response amplitude of the 16-day-old subjects on the two types of test trials. This developmental pattern of results is similar to that reported by Campbell and Ampuero (1985) and Richardson et al. (1993) for conditioned changes in HR to the same CS. In those studies, animals did not show conditioned HR changes to the tone until 21-23 days of age.
When fear-potentiated startle was observed, acquisition in the paired subjects proceeded rapidly, as indicated by the differences between the paired and unpaired groups' responding during the training episode. The unpaired animals showed rapid habituation of the response to the startle pulse. The paired animals, however, showed little habituation, responding at a consistently higher level than the unpaired groups. The conditioning procedure appeared to mask habituation of the response to the startle stimulus in the paired subjects. By comparing the magnitude of the response of the paired groups on the noise-alone test trials (see Figure 1) with the level of habituated responding by the unpaired groups, one can see that the magnitude of these responses were comparable. This indicates that the response to the startle pulse of the paired groups did habituate during training, but by preceding the startle pulse with the CS on each conditioning trial, the high level of responding was maintained. Leaton and Cranney (1990) also observed this effect, noting that "the response-enhancing effects of conditioned fear can mask the response-reducing effects of habituation" (p. 279).
The failure to obtain fear-potentiated startle in the 16-dayolds could have been due to the absence of freezing to the CS (Leaton & Borszcz, 1985). There is some evidence suggesting that young rats may not express fear conditioning in terms of freezing behavior immediately after training but that they do after 24 hr (Moye & Rudy, 1985, 1987; Mellon et al., 1991), so it is possible that 16-day-olds would show fear-potentiated startle if they were tested 24 hr after the initial conditioning procedure. Interestingly, adult rats are typically tested for potentiated startle at least 24 hr after fear training (Campeau & Davis, 1992; Davis, 1992a, 1992b; Leaton & Borszcz, 1985; Leaton & Cranney, 1990).
The purpose of Experiment IB was to condition 16- and 75-day-old animals with the same procedures used in Experiment 1A. However, the rats were tested for potentiated startle 24 hr later rather than immediately after completion of the training sequence.
Figure 3. Mean (and standard error of the mean) startle responses (in millivolts) of 16- and 75-day-old subjects of Experiment IB. Rats were given paired or unpaired presentations of a tone conditioned stimulus (CS) with a startle pulse unconditioned stimulus during the acquisition phase of the procedure. These test data were obtained 24 hr after training. Subjects were given 20 startle pulse-alone trials followed by 6 test trials in random order. Three of these trials consisted of the startle pulse presented alone (noise-alone trials), and the other 3 consisted of the startle pulse preceded by the tone CS (tonenoise trials).
Experiment IB: Method
Subjects. The subjects were 16 naive rats that were 16 days old (±1 day) and 20 that were 75 days old (±15 days). They were maintained as described in Experiment 1A.
Procedure. Training was the same as in Experiment 1A. Following the four tone-alone habituation trials, rats were given 20 paired or explicitly unpaired CS-US presentations. Following training, they were returned to the home cage for a 24-hr retention interval.
The test procedures were based on those used by Davis (e.g., Campeau & Davis, 1992) to assess fear-potentiated startle. Rats were first given 20 unsignaled startle pulse presentations with 30-60-s ISIs to partially habituate the startle response to the 130-dB acoustic stimulus. Immediately following these habituation trials, animals were given six test trials, three consisting of the startle pulse alone (noise alone) and three consisting of the startle pulse preceded by the tone CS (tone-noise). These test trials were exactly as those described in Experiment 1A.
Experiment IB: Results
Test data. A 2 (group) x 2 (trial type) mixed ANOVA conducted on the test data obtained from the 16-day-olds yielded nonsignificant effects (all Fs < 1.0). Regardless of training experience and test trial type, all rats responded equally to the startle pulse. The ANOVA conducted on the test data obtained from the adult subjects yielded a significant main effect of trial type, F(l, 18) = 16.85,p < .01, as well as a significant interaction between group and trial type, F(l, 18) = 5.98, p < .05. The effect of group approached significance, F(l, 18) = 3.99,p < .06. Post hoc comparisons confirmed that the paired subjects responded more on the tone-noise trials than on the noise-alone trials, whereas the unpaired animals responded similarly on both test trial types. The magnitude of the differences between the paired and unpaired groups were similar to those observed immediately after training (Experiment 1A). These data are presented in Figure 3.
Acquisition data (Day 1). The 2 (group) x 10 (trial block) mixed ANOVA conducted on the acquisition data obtained from the 16-day-old subjects revealed a significant main effect of trial block, F(9,126) = 5.89, p < .01, but no effect of either group or the Group x Trial Block interaction (Fs < 1.0). Regardless of group, all rats showed habituation of the startle response during the training session. Analysis of the acquisition data obtained from the adult subjects yielded a significant main effect of group, F(l, 18) = 6.19,p < .05. The main effect of trial block and the Group x Trial Block interaction were both nonsignificant, Fs(9, 162) = 1.50 and 1.63, respectively. The response of the paired group to the startle pulse was consistently larger than that of the unpaired group across the training session. These data were similar to those reported in Experiment 1A with identical training procedures.
Habituation (Day 2). The 2 (group) x 10 (trial block) mixed ANOVAs conducted on the responses to the startle stimulus during the habituation session for each age both yielded significant main effects of trial block for 16-day-olds, F(9,126) = 4.88,p < .01, and adults, F(9,162) = 8A2,p < .01. Startle response amplitudes were high at the beginning of the session for all rats and then habituated steadily across trials. There was no effect of prior training on the initial level of startle responding or on the rate of habituation of the startle response. The mean startle responses on Trial Blocks 1 and 10, collapsed across group, were 106.3 and 61.6 for the pups and 137.7 and 59.7 for the adults.
Experiment IB: Discussion
These results, combined with those of Experiment 1A, show that 16-day-olds did not exhibit fear-potentiated startle, either during the conditioning session, immediately following conditioning, or after a 24-hr retention interval. The results of Experiments 1A and IB also suggest that the neural mecha
Figure 4. Mean (and standard error of the mean) startle responses (in millivolts) of 23-, 30-, and 75-day-old subjects of Experiment 2. Rats were given paired or explicitly unpaired presentations of a 10-s flashing light conditioned stimulus (CS) with the startle pulse and were immediately tested for fear-potentiated startle responding. The noise-alone test trials consisted of presenting the startle pulse unsignaled, whereas the light-noise trials consisted of the startle pulse preceded by the 10-s light CS.
nisms necessary to establish fear-potentiated startle to an 0.25 s on and 0.25 s off. Additionally, the houselight inside the IAC was auditory CS do not mature until relatively late in development. off throughout all training and testing. Moreover, there is a remarkable similarity between the age at Procedure. The procedure for training the rats was similar to that used in Experiment 1A. Animals of each age were randomly assigned which fear-potentiated startle to a 1600-Hz tone first occurs to one of two groups (paired or unpaired; all .s = 10). Paired subjects and the earliest age that conditioned changes in heart rate to were placed into the restraining cage in the IAC and remained there that same stimulus can be established (Campbell & Ampuero, for a 15-min period of adaptation. Next, these subjects received four 1985; Richardson et al., 1993). This suggests that there is a light-alone habituation trials followed by 20 pairings of the 10-s close parallel between the emergence of fear-potentiated flashing light and the startle pulse. The intertrial intervals ranged from startle and conditioned autonomic activity in the developing 60 to 120 s. Unpaired animals were treated similarly, except that the rat. light CSs and startle pulse USs were presented in an explicitly unpaired fashion. The ISIs in the unpaired condition varied between 30 and 60 s. Animals were trained and tested individually.
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